摘要:

AbstractUptake of [U‐14C]sorbitol was studied in astroglia‐rich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 μMand 400 mM.Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cy‐tochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, andn‐hexanol, all compound that alter the properties of biological membranes, and the sulfhydryl reagentp‐chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel‐li

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11755.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractA lysosomal preparation, obtained from brain ho‐mogenate of 17‐day‐old C57BL mice by centrifugation on a self‐generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40–120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GDla and fluorimetrically with 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid (MUB‐NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane‐bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma Membrane‐bound enzyme. The apparentKmvalues for GDl a and MUB‐NeuAc were 1.5 × 10‐5and 4.2 × 10‐5M, respectively, for the lysosomal enzyme and 2.7 × 10‐4and 6.3 × 10‐5Mfor the plasma membrane‐bound one. Triton ×‐ 100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane‐bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane‐bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane‐bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers. It is thus evident that the sialidase present in the lysosomal fraction is an authentic lysosomal enzyme distinct and different from the sialidase contained in the plasma membrane. The lysosomal sialidase affected other ganglio‐sides, like GDlb and GM3. These data constitute the first direct evidence for the presence in brain lysosomes of a sialidase activity on gangliosides and contribute to a better knowled

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11756.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractDepoiarisation of [3H]inosito]‐prelabejled slices of rat cerebral cortex with elevated extracellular Kr induced a rapid and marked increase in inositol polyphosphate accumulation. Addition of the muscarinic antagonist atropine (10 μM) markedly inhibited the K+‐induced accumulation of inositol tetrakisphosphate (InsP4), with only a slight reduction in stimulated inositol bis‐ and trisphosphate levels. Inhibitory effects on InsP4were noted at the earliest time period measured (30 s) and suggested the involvement of released endogenous acetylcholine in part of the response. The atropine‐insensitive component of depoiarisation did not! appear to be secondary to release of noradrenaline, histamine, or 5‐hydroxytryptamine, because addition of prazosip, mepyr‐amine, or ketanserin was without effect on the K+ response. Furthermore, secretion of a neuropeptide that could stimulate phosphoinositide hydrolysis was unlikely, because the peptidase inhibitor bacitracin was also without effect. The results suggest that endogenous acetylcholine can stimulate phosphoinositide metabolism by interacting with muscarinic receptors and that this is particularly evident on InsP4accumulation. Atropine‐insensitive responses may be secondary to Ca2+entry via voltage‐se

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11757.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractSerotonin has no obvious effect on basal cyclic AMP levels but reduces the forskolin‐, isoproterenol‐, and vasoactive intestinal peptide‐induced stimulation of cyclic AMP levels in a dose‐dependent manner. Serotonergic, cholinergic, muscarinic, α‐adrenergic, and dopaminergic antagonists have no effect on the serotonin response. Topical Application of a serotonin/pargyline solution to the living eye causes desen‐sitisation of the serotonin response in the iris‐ciliary body, an observation confirming the presence of specific serotonergic receptors linked to adenylate cyclase. The 5‐HT1A[5‐hy‐droxytryptamine (serotonin) type 1A] receptor agonists 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin and bujspirone mimic the serotonin response in reducing the forskolin‐stimulated cyclic AMP levels, as do the indole derivatives 5‐methoxy‐ tryptamine, 5‐hydroxtryptophan, and tryptamine. However, the ineffectiveness of the 5‐HT1Aagonist ipsapirone and the inability of spiroxatrine to block the serotonin response show that classical 5‐HT1Areceptors are not involved. The serotonin response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophylline, which indicates the involvement of an inhibitory guanine regulatory protein in the coupling of the serotonin recept

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11758.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractThe total activity of superoxide dismutase (SOD) and cytosolic and particulate activity of SOD in human substantia nigra and cerebellum were measured by a spectrophotometric method based on the ability of SOD to inhibit the autoxidation of adrenaline. The cystosolic and particulate isoenzymes of SOD were differentiated by the inclusion of potassium cyanide which selectively inhibits cytosolic copper/zinc‐dependent SOD activity. In autopsied human brains, there was no difference in total SOD) activity, or the activity of SOD in cytosol in substantia nigra jof patients dying with Parkinson's disease compared to age‐matched controls. However, the activity of the particulate form of SOD was higher in the parkinsonian substantia nigra compared to control tissue. In the cerebellum there was no difference in the total, cytosolic, or particulate activity of SOD between parkinsonian patients and age‐matched controls. Increased activity of SOD in particulate fraction may be a protective response to elevated levels of toxic free radicals in the parkinsonian substantia nigra. Alternatively, increased SOD activity may induce cell death through the accumulation of hydrogen per

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11759.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractThe novelN‐methyl‐D‐aspartate recjeptor channel ligand (+)‐[3H]5‐methyl‐J0, l1‐dihydro‐5H‐dibenzo[a, d]‐cyclohepten‐5, 10‐imine maleate ([3H]MK‐801) has been utilized to label this receptor in human brain tissiie. Characteristics of [3H]MK‐801 binding to well‐washeq membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of the and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding Was observed in the frontal cortex under glutamate‐ and glycine‐stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine‐insensitive g

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11760.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractThe ganglioside composition in meningioma specimens from 20 patients was analyzed to find potential me‐ningioma‐associated structures. The characterization was performed by immunological staining with specific monoclonal antibodies to gangJioside antigens and fast atom bombardment‐mass spectrometry. The major gangliosides were GM3 and GD3, and most of the meningioma specimens could be divided into a “GM3‐rich” or a “GD3‐rich” group. Gangliosides of the gangliotetraose series were represented by GM1, GD1 a, GDlb, and GT1b, which were found in minor amounts in all the specimens. The ratios of GM1/ GD1a and GD1a/GDlb differed from that in normal brain, and therefore existence of this series could not be explained by contamination with brain material. Ganglioside 3′‐isoLM1, found in human malignant glioma, could not be detected in an

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11761.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstractβ‐N‐OxalyJamino‐L‐aJanine (L‐BOAA) is a nonprotein excitatory amino acid present in the seed ofLathyrus sativusL. This excitotoxin has been characterized as the causative agent of human neurolathyrism; an upper motor neuron disease producing corticospinal dysfunction from excessive consumption of the lathyrus pea. Previous behavioral, tissue‐culture, and in vitro receptor binding investigations revealed that L‐BOAA might mediate acute neurotoxicity through quisqualate (QA)‐preferring glutamate receptors. The present study demonstrates the stereospecific action of L‐BOAA on glutamate receptor binding in whole mouse brain synaptic membranes. L‐BOAA was most active in displacing thiocyanate (KSCN)‐sensitive specific tritiated (RS)‐α‐3‐hy‐droxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) binding (i.e., QA receptor) (Ki= 0.76 μM) with a rank‐order potency of QA>kainate>N‐methyl‐D‐aspartate (NMDA). By contrast, the nonneurotoxic D‐BOAA isomer (100 μM) was essentially inactive in displacing radioligands for glutamate receptors, except the NMDA site, where it was equipotent with L‐BOAA. Scatchard analysis of L‐BOAA displacement of specific [3H]AMPA binding indicated competitive antagonism (KD: control, 135 nM; L‐BOAA, 265 nM) without a significant change in QA‐receptor density, and Hill plots yielded coefficients approaching unity. Differential L‐BOAA concentration‐dependent decreases in specific [3H]AMPA binding were observed in synaptic membranes, indicating that the neurotoxin was more potent in displacing specific binding from frontal cortex membranes, followed by that for corpus striatum, hippocampus, cerebellum, and spinal cord. Comparative experiments for inhibition of specific [3H]‐AMPA binding in cortex revealed that L‐BOAA was approximately five‐ and 10‐fold less potent than QA and AMPA, respectively, but twofold greater than the endogenous neurotransmitter, glutamate. Parallel studies with KSCN‐treated spinal cord synaptic membranes (compared to cortex controls) indicated that specific [3H]AMPA binding in untreated tissue was (a) approximately 5% of cortex and pharmacologically distinct and (b) differentially sensitive to L‐BOAA and stimulation of binding by KSCN. These data suggest that low levels of L‐BOAA in vivo might initially exert excitotoxicity through sensitive cortex QA‐preferring neurons while eliciting little interference with QA sites in the spinal cord. Although the precise role(s) of the excitatory amino acid synapse in neurodegenerative diseases remains unclear, the cortex QA receptor could be preferentially sensitive to L‐BOAA attack and might represent the initial molecular rec

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11762.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractCrude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl‐N‐acetylgalactosalminyl‐(N‐acetyl‐neuraminyl)‐galactosylglucosyl ceramide (GMl) on high‐affinity binding of L‐[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40–50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with3H‐labeled GM1 proved trypsin‐stable insertion of GM1 into SPM, with a maximum incorporation pf four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+‐stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 μl/mg of protein, in ganglioside‐treated as well as in control membranes. From the data, it is concluded that a special. Na+‐ and C1‐independent form of glutamate transport into resealed membrane vesicles is stimulated by gangli

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11763.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

AbstractGlycine potentiates stimulation of inositol phospholipid hydrolysis by glutamate andN‐methyl‐D‐aspartate, but not by quisqualate or carbamylcholine, in primary cultures of cerebellar granule cells. This potentiation occurs in the absence of extracellular Mg2+, but is more evident when stimulation of inositol phospholipid hydrolysis byN‐methyl‐D‐aspartate is measured in the presence of 1 mMMg2+. The action of glycine is not antagonized by strychnine. These results suggest that glycine acts as a positive modulator of signal transduction at a specific class ofN‐methyl‐D‐aspartate‐sensitive glutamate receptors coupled to inositol phospholipid hydrolysis in cereb

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb11764.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY